Use of il-12 to increase survival

ABSTRACT

The present invention provides methods for increasing survival in a subject, and/or preserving bone marrow function, and/or promoting hematopoietic recovery or restoration. The methods include administering a dose of IL-12 to the subject following an acute exposure to non-therapeutic whole body ionizing radiation. Formulations and kits are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present invention claims priority to U.S. Provisional PatentApplication No. 61/125,508, filed Apr. 24, 2008, the teachings of whichare hereby incorporated by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with US Government support under contract numberBAA-BARDA-08-08 awarded by the Biomedical Advanced Research andDevelopment Authority, within the Department of Health and HumanServices. The US Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The hematological effects of acute, high dose total body irradiation(TBI) can lead to death without supportive care, such as hematopoietictransplant, transfusions and other supportive care measures. However,such supportive measures cannot be readily administered to a potentiallylarge number of victims of high dose radiation exposure following anuclear accident or direct acts of terrorism. If such disasters were tooccur, military personnel and civilians alike would be left in greatjeopardy. One of the deleterious effects of high dose radiation is theinduction of a hematopoietic syndrome. To counteract the potentiallylethal effects of hematopoietic syndrome, effective remedial drugs thatcan be quickly distributed shortly after the radiation incident are ingreat need.

Currently, there are no available drugs that are effective in increasingsurvival and regenerating hematopoiesis. Moreover, in the face of aradiation-related disaster or act of terrorism, the immediatedistribution of such effective drugs to military personnel or civilians,if these were to be available, would not be practically possible. It isanticipated that a lag time of at least several hours, and perhaps 24hours or longer, would be necessary to distribute such drugs to thescene of such a radiation disaster or act of terrorism. Thus, it iscritically important that effective drugs that can be used to increasesurvival and regenerate hematopoiesis exhibit efficacy at protractedtime intervals following acute exposure to ionizing radiation.

Several studies performed in the mid 80's suggested that proinflammatorycytokines could confer radioprotection when administered prior to lethaldoses of radiation (Neta R. et al., Lymphokine Res. 1986; 5 Suppl1:S105-10; Neta R. et al., J Immunol. 1986 Apr. 1; 136(7):2483-5;Schwartz G. N. et al., Immunopharmacol Immunotoxicol. 1987;9(2-3):371-89). However, it was recognized by several later studies thatthe use of IL-12 for prophylactic and therapeutic treatments of lethalirradiation suffered from significant drawbacks. One such adverse effectwas that IL-12 administered at a high dose of 1000 ng per mouseradiosensitized, rather than radioprotected, the gastrointestinal tract,resulting in lethal gastrointestinal syndrome in irradiated mice (NetaR. et al., J Immunol. 1994 Nov. 1; 153(9):4230-7). This work led to theconclusion that IL-12 sensitized the intestinal tract at levelsnecessary for protection of bone marrow cells (Neta R., Environ HealthPerspect. 1997 Dec.; 105 Suppl 6:1463-5).

Consistent with these findings, Hixon et al. (Hixon et al., Biol BloodMarrow Transplant. 2002; 8(6):316-25) showed that repeatedadministration of 500 ng IL-12 to BALBc mice who received bone marrowtransplants following lethal whole body irradiation, resulted in acutelethal toxicity within 4 to 6 days. In contrast, Hixon et al.demonstrate that under identical conditions, BALBc mice that did notreceive IL-12 administration recovered 100% of the time.

In the event of a nuclear event, whether accidental or malicious,pre-administration of drugs that promote survival is simply notpossible. For example, in the event of a nuclear disaster, where largenumbers of people and animals may require therapeutic administration,sufficient time will be required for the large-scale distribution ofradiation treatments. Methods are needed for treatment that areeffective when administered at protracted times following acute exposureto whole body ionizing radiation.

As such, there remains a need in the art for drugs that are effective inincreasing survival and regenerating hematopoiesis when administrated atprotracted times following acute exposure to whole body ionizingradiation. The present invention satisfies these and other needs byproviding methods of treating a subject following an acute exposure tonon-therapeutic whole body ionizing radiation via administration ofIL-12 at protracted timepoints.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the use of Interleukin-12 (IL-12) forincreasing survival and promoting hematopoietic recovery following acuteexposure to non-therapeutic ionizing radiation. Use of the methods ofthe present invention will increase the number of survivors from aradiation-related disaster, such as a terrorist attack with a dirtybomb.

In one aspect, the present invention provides methods for increasingsurvival, and/or preserving bone marrow function, and/or promotinghematopoietic recovery or restoration in a subject following acuteexposure to non-therapeutic, acute whole body ionizing radiation,comprising the administration of a therapeutically effective dose ofIL-12.

In a related aspect, the present invention provides pharmaceuticallyacceptable formulations of IL-12 for increasing survival, and/orpreserving bone marrow function, and/or promoting hematopoietic recoveryor restoration in a subject following acute exposure to non-therapeuticwhole body ionizing radiation.

In another aspect, the present invention provides kits useful forincreasing survival, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration in a subject followingacute exposure to non-therapeutic whole body ionizing radiation,comprising one or more therapeutically effective dose of IL-12.

Further aspects, objects, and advantages of the invention will becomeapparent upon consideration of the detailed description and figures thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-C) shows that IL-12 facilitates multilineage hematopoieticrecovery at various time points of administration and dosages, and evenfacilitates significant recovery of all blood groups at 3 hours postradiation (9 Gy). The blood recovery of neutrophils (1A), red bloodcells (1B) and platelets (1C) are shown in the graph. The normalthreshold count level is depicted for the different blood cell groups(dotted horizontal line). The depicted treatment groups are as follows:IL-12 (200 ng; 30 μg/m²) administered 3 hrs after radiation (♦), IL-12(100 ng; 15 μg/m²) administered 24 hours before radiation (), IL-12administered 24 hours before and 2 hr after radiation (100 ng pre/50 ngpost; 15 μg/m² pre/7.5 μg/m² post) (▪), and IL-12 administered 24 hoursbefore and 2 hr after radiation (100 ng pre/100 ng post; 15 μg/m²pre/185 μg/m² post) (▴). No control mice remained at 16 days postradiation for blood analysis. Bactrim was removed at 48 days.

FIG. 2 shows survival effects of IL-12 when administered at 6 hoursafter a lethal dose of radiation (LD_(100/10)) and subcutaneousinjection. Female mice C57BL/6 (9 weeks) were used for this experiment(5-6 mice per group).

FIG. 3 shows the results of one aspect of the invention as aKaplan-Meier plot. Group 1 received vehicle (phosphate buffered saline(PBS)) and Group 2 received an initial dose of IL-12 (120 ng; 18 μg/m²)at 6 hours after radiation, followed by one subsequent dose of IL-12(100 ng; 15 μg/m² at 3 days post radiation). No antibiotic support wasadministered. The radiation dose was 8 Gy (0.9 Gy/min); P=0.001 viaMantel Chi-square analysis. Mice did not receive antibiotic support.

FIG. 4 shows remarkable survival effects when administered at 6 hoursafter a lethal dose of radiation (LD_(100/21)) and subcutaneousinjection in male mice. C57BL/6 (14 weeks) were used for this experiment(5-6 mice per group). Mice were first exposed to a non-lethal dose ofradiation at 7 Gy. At 6 hours after radiation, mice were either treatedwith PBS (Group 1) or IL-12 (Group 2 (150 ng; 22.5 μg/m²). All micesurvived the 7 Gy dose of radiation. On the next round of radiation, allmice received an 8 Gy radiation dose (0.9 Gy/min). On the second roundof radiation, Group 3 received PBS and Group 2 again received the samedose of IL-12 (150 ng; 22.5 μg/m²) that was administered in the firstround of radiation, followed by a subsequent dose (100 ng; 15 μg/m²) 48hrs after the initial dose at 6 hr post radiation.

FIG. 5 shows a Kaplan-Meier plot from the second round of radiation. Noantibiotic support was administered; P<0.05 via Mantel Chi-squareanalysis.

FIG. 6 shows survival effects as a result of IL-12 administration at 24hours after a lethal dose of radiation (LD_(90/27)) and subcutaneousinjection. Female mice C57BL/6 (9 weeks) were used for this experiment(7-8 mice per group). Group 1 received vehicle (phosphate bufferedsaline (PBS)) and Group 2 received an initial dose of IL-12 (120 ng; 18μg/m²) at 24 hours after radiation, followed by one subsequent dose ofIL-12 (100 ng; 15 μg/m² at 3 days post radiation). No antibiotic supportwas administered. The radiation dose was 8 Gy (0.9 Gy/min); P=0.001 viaMantel Chi-square analysis.

FIG. 7 shows a Kaplan-Meier plot at 27 days in the experimentaltimeline. No antibiotic support was used. P=0.005 via Mantel Chi-squareanalysis.

FIG. 8 (A-B) shows the results of female C57BL/6 mice (10 per group),which were irradiated with an LD₈₀ dose of acute irradiation. The micewere then administered the indicated amounts of IL-12 as either (A) adouble dose at 24 hours and 72 hours post irradiation, or (B) a singledose at 24 hour post irradiation. The survival rates for each of thegroups are shown.

FIG. 9 shows the results of female C57BL/6 mice (10 per group), whichwere irradiated with an LD₈₀ dose of acute irradiation. The mice werethen administered the indicated amounts of IL-12 as either (A) a doubledose at 24 hours and 72 hours post irradiation, or (B) a single dose at24 hour post irradiation. The average weight of the surviving mice areplotted as a function of time in days.

FIG. 10 illustrates a stratified K-M plot for double dose experimentsperformed with female C57BL/6 mice (10 per group). Briefly, the micewere irradiated with an LD₈₀ dose of acute irradiation followed byadministration of a double dose of IL-12, as described in example 6, at24 and 72 hours post irradiation. Survival analysis did not indicate asignificant overall effect of IL-12 dose on survival (p<0.59;Tarone-Ware test).

FIG. 11 illustrates a K-M plot of survival for group 1 (0 ng IL-12,vehicle control; (▴)) and group 7 (300 ng IL-12; 45 μg/m²; ()) femaleC57BL/6 mice irradiated with an LD₈₀ dose of acute irradiation followedby administration of IL-12 at 24 and 72 hours post irradiation. Survivalanalysis reveals that administration of 300 ng (45 μg/m²) IL-12 at both24 and 72 hours post irradiation resulted in a significant increase insurvival (p<0.03; Tarone-Ware and Mantel-Cox tests).

FIG. 12 illustrates a stratified K-M plot for single dose experimentsperformed with female C57BL/6 mice (10 per group). Briefly, the micewere irradiated with an LD₈₀ dose of acute irradiation followed byadministration of a single dose of IL-12, as described in example 6, at24 hours post irradiation. Survival analysis indicates a significantoverall effect of IL-12 dose on survival (p<0.02; Tarone-Ware test).

FIG. 13 illustrates a K-M plot of survival for group 1 (0 ng IL-12,vehicle control; (▴)), group 2 (40 ng IL-12; 6 μg/m² (▪)), and group 7(300 ng IL-12; 45 μg/m² ()) female C57BL/6 mice irradiated with an LD₈₀dose of acute irradiation followed by administration of IL-12 at 24hours post irradiation. Survival analysis reveals that administration of40 ng (6 μg/m²) and 300 ng (45 μg/m²) IL-12 at 24 hours post irradiationresulted in a significant increase in survival (p<0.001; Tarone-Waretest).

FIG. 14 illustrates a dose response curves for single doseadministration of IL-12 24 hours after irradiation. Female C57BL/6 miceirradiated with an LD₈₀ dose of acute irradiation followed byadministration of IL-12 at 24 hours post irradiation, as in example 6.Survival time and percent weight loss are plotted as a function ofadministered IL-12 dose.

FIG. 15 illustrates a survival curve for female C57BL/6 mice irradiatedwith various acute doses of ionizing radiation.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Interleukin-12 (IL-12)” as used herein includes any recombinant IL-12molecule that yields at least one of the properties disclosed herein,including native IL-12 molecules, variant IL-12 molecules and covalentlymodified IL-12 molecules, now known or to be developed in the future,produced in any manner known in the art now or to be developed in thefuture. Generally, the amino acid sequence of the IL-12 molecule used inembodiments of the invention is the canonical human sequence related toIL-12p70. IL-12 comprises two subunits, IL-12A (p35) and IL-12 B (p40)Polymorphisms, however, are known to exist for IL-12, especially in thep35 subunit. In particular, a known polymorphism can exist at amino acid247 of the p35 human subunit, where methionine is replaced by threonine.Still other embodiments of the invention include IL-12 molecules wherethe native amino acid sequence of IL-12 is altered from the nativesequence, but the IL-12 molecule functions to yield the properties ofIL-12 that are disclosed herein. Alterations from the native,species-specific amino acid sequence of IL-12 include changes in theprimary sequence of IL-12 and encompass deletions and additions to theprimary amino acid sequence to yield variant IL-12 molecules. An exampleof a highly derivatized IL-12 molecule is the redesigned IL-12 moleculeproduced by Maxygen, Inc. (Leong S R, et al., Proc Natl Acad Sci USA.2003 Feb. 4; 100(3):1163-8.), where the variant IL-12 molecule isproduced by a DNA shuffling method. Also included are modified IL-12molecules included in the methods of invention, such as covalentmodifications to the IL-12 molecule that increase its shelf life,half-life, potency, solubility, delivery, etc., additions ofpolyethylene glycol groups, polypropylene glycol, etc., in the mannerset forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, each of which is hereby incorporated byreference. One type of covalent modification of the IL-12 molecule isintroduced into the molecule by reacting targeted amino acid residues ofthe IL-12 polypeptide with an organic derivatizing agent that is capableof reacting with selected side chains or the N- or C-terminal residuesof the IL-12 polypeptide. Other IL-12 variants included in the presentinvention are those where the canonical sequence has been altered toincrease the glycosylation pattern of the resultant IL-12 molecule, ascompared with the native, non-altered IL-12. This method has been usedto generate second generation molecules of erythropoietin, referred toas Aranesp. Both native sequence IL-12 and amino acid sequence variantsof IL-12 may be covalently modified. Also as referred to herein, theIL-12 molecule can be produced by various methods known in the art,including recombinant methods. Since it is often difficult to predict inadvance the characteristics of a variant IL-12 polypeptide, it will beappreciated that some screening of the recovered variant will be neededto select the optimal variant. A preferred method of assessing a changein the properties of variant IL-12 molecules is via the lethalirradiation rescue protocol disclosed below. Other potentialmodifications of protein or polypeptide properties such as redox orthermal stability, hydrophobicity, susceptibility to proteolyticdegradation, or the tendency to aggregate with carriers or intomultimers are assayed by methods well known in the art.

“Acute Radiation Syndrome” in humans as used herein includes an acuteradiation exposure of 2 Gy or greater.

“Hematopoietic Syndrome” as used herein includes damage to the bonemarrow compartment which results in pancytopenia, i.e., a deficiency inperipheral blood cell counts for all blood cell types, namely whiteblood cells, red blood cells and platelets. Hematopoietic Syndrome alsorefers to loss of hematopoietic progenitor and stem cells in the bonemarrow compartment.

“Survival” as used herein includes an increase in survival that can bemeasured in non-human species as compared to control groups, such asmice or non-human primates.

“Hematopoietic Recovery” as used herein includes early recovery ofperipheral blood cell counts for white blood cells, red blood cells andplatelets, as compared to control groups and as measured in non-humanspecies, such as mice or non-human primates.

“Preservation of bone marrow function” as used herein includes earlyrecovery of cellularity or colony forming units in the bone marrowcompartment, or any other measure of bone marrow function, as comparedto control groups and as measured in non-human species, such as mice andnon-human primates.

A “therapeutically effective amount or dose” or “sufficient amount ordose” as used herein includes a dose that produces effects for which itis administered, for example, a dose sufficient for increasing survival,and/or preserving bone marrow function, and/or promoting hematopoieticrecovery or restoration in a subject that has been exposed to an acutedose of whole body ionizing radiation. The exact dose will depend on thepurpose of the treatment, the timing of the IL-12 administration,certain characteristics of the subject to be treated, the total amountor timing of irradiation, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Pickar, Dosage Calculations(1999); and Remington: The Science and Practice of Pharmacy, 20thEdition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

Generally, a dose of a therapeutic agent, according to the methods andcompositions of the present invention, can be expressed in terms of thetotal amount of drug to be administered (i.e., ng, μg, or mg).Preferably, the dose can be expressed as a ratio of drug to beadministered to weight or surface area of subject receiving theadministration (i.e., ng/kg, μg/kg, ng/m², or μg/m²). When referring toa dose in terms of the mass to be administered per mass of subject(i.e., ng/kg), it will be understood that doses are not equivalentbetween different animals, and thus conversion factors will need to beused to ensure that one animal receives the same dose equivalent asanother animal. Suitable factors for the conversion of a mouse “doseequivalent” to a “dose equivalent” of a different animal are given inthe look-up table below. Thus, in a preferred embodiment, doses aregiven in terms of mass to surface area (i.e., ng/m², or μg/m²), whichare equivalent for all animals. The following basic conversion factorscan be used to convert ng/kg to ng/m²: mouse=3.0, hamster=4.1, rat=6.0,guinea pig=7.7, human=38.0 (Cancer Chemother Repts 50(4):219(1966)).

TABLE 1 Conversion factors and equivalent doses for several animals.Weight Total Dose Dose Dose Conversion Species (kg) (ng) (ng/kg) (ng/m²)Factor Human 65 25655.82 394.7 15000 0.0794 Mouse 0.02 99.47 4973.4415000 1.0000 Hamster 0.03 130.2 4339.87 15000 0.8726 Rat 0.15 381.122540.8 15000 0.5109 Guinea Pig 1.00 1335 1335 15000 0.2684 Rabbit 22381.1 1190.55 15000 0.2394 Cat 2.5 2956.44 1182.57 15000 0.2378 Monkey3 3681.75 1227.25 15000 0.2468 Dog 8 6720 840 15000 0.1689

As used herein, the term “intermediate dose” includes doses between arange of about 15 μg/m² and about 75 μg/m², or between a range of about20 μg/m² and about 75 jμg/m², between a range of about 22.5 μg/m² andabout 67.5 μg/m², between a range of about 22.5 μg/m² and about 52.5μg/m², between a range of about 30 μg/m² and about 60 μg/m², between arange of about 37.5 μg/m² and about 52.5 μg/m², and all doses and rangesin-between.

As used herein, the term “low dose” includes doses less than about 15μg/m², or less than about 14 μg/m², or less than about 13 μg/m², 12μg/m², 11 μg/m², 10 μg/m², 9 μg/m², 8 μg/m², 7 μg/m², 6 μg/m², 5 μg/m²,4 μg/m², 3 μg/m², 2 μg/m², 1 μg/m², or less than about 900 ng/m², 800ng/m², 700 ng/m², 600 ng/m², 500 ng/m², 400 ng/m², 300 ng/m², 200 ng/m²,or 100 ng/m². In certain embodiments, a low dose includes an ultralowdose.

As used herein, the term “ultralow dose” includes doses less than about3 μg/m², 2 μg/m², 1 μg/m², or less than about 900 ng/m², 800 ng/m², 700ng/m², 600 ng/m², 500 ng/m², 400 ng/m², 300 ng/m², 200 ng/m², or 100ng/m².

II. Embodiments

In one aspect, the present invention is based on the surprisingdiscovery that Interleukin-12 (IL-12) increases the survival of subjectsexposed to lethal and sub-lethal acute doses of non-therapeutic wholebody ionizing radiation. Significantly, it was found that administrationof IL-12 following acute doses of ionizing radiation resulted in theregeneration of hematopoietic activity and peripheral blood cell countsin lethally irradiated mice, yielding complete hematopoietic recoverywithout the use of transplanted cells.

Advantageously, it was found that IL-12 has the unique and remarkableproperty of being able to confer survival on lethally irradiated mammalswhen administered at protracted time points after radiation.Specifically, it was found that IL-12 can rescue about 90-100% ofmammals when administered at 6 hours or 24 hours after radiation.

In one aspect, a single dose of IL-12 is sufficient to confersignificant survival, bone marrow preservation, and promotion ofhematopoietic recovery. In other aspects, IL-12 may be administered inmore than one dose such as 2, 3, 4, 5 or more doses.

Accordingly, in one aspect, the present invention provides a method forincreasing survival in a subject, and/or preserving bone marrowfunction, and/or promoting hematopoietic recovery or restorationcomprising the administration of a dose of IL-12 to the subjectfollowing an acute exposure to non-therapeutic whole body ionizingradiation. In one embodiment, the dose of IL-12 is less than about 100μg/m². In one embodiment, the dose of IL-12 is less than about 75 μg/m².In another embodiment, the low dose can be between about 1 μg/m² andabout 100 μg/m², such as 1 μg/m², 5 μg/m², 10 μg/m², 15 μg/m², 20 μg/m²,25 μg/m², 30 μg/m², 35 μg/m², 40 μg/m², 45 μg/m², 50 μg/m², 55 μg/m², 60μg/m², 65 μg/m², 70 μg/m², 75 μg/m², 80 μg/m², 85 μg/m², 90 μg/m², 95μg/m² and 100 μg/m² and all doses in-between. In a particularembodiment, the preferred dose of IL-12 is less than about 75 μg/m². Inone embodiment, the dose of IL-12 is either between a range from about100 μg/m² to about 18 μg/m² or between a range from about 15 μg/m² toabout 1 μg/m².

In certain embodiments, the IL-12 is a mammalian IL-12, recombinantmammalian IL-12, murine IL-12 (mIL-12), recombinant murine IL-12(rmIL-12), human IL-12 (hIL-12), recombinant human IL-12 (rhIL-12),canine IL-12 or rIL-12, feline IL-12 or rIL-12, bovine IL-12 or rIL-12,equine IL-12 or rIL-12, or biologically active variants or fragmentsthereof. In one specific embodiment, the rhIL-12 is HemaMax™(Neumedicines Inc.). In certain embodiments, the IL-12 can be modifiedin a fashion so as to reduce the immunogenicity of the protein afteradministration to a subject. Methods of reducing the immunogenicity of aprotein are well known in the art and include, for example, modifyingthe protein with one or water soluble polymers, such as a PEG, a PEO, acarbohydrate, a polysialic acid, and the like.

In another aspect, the present invention provides methods for increasingsurvival in a subject, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration comprising theadministration of an intermediate dose of IL-12 to the subject followingan acute exposure to non-therapeutic whole body ionizing radiation. Inone embodiment, the intermediate dose of IL-12 is between a range ofabout 15 μg/m² and about 100 μg/m², or between a range of about 20 μg/m²and about 75 μg/m², between a range of about 22.5 μg/m² and about 67.5μg/m², between a range of about 22.5 μg/m² and about 52.5 μg/m², betweena range of about 30 μg/m² and about 60 μg/m², between a range of about37.5 μg/m² and about 52.5 μg/m², and all doses and ranges in-between. Ina particular embodiment, the preferred intermediate dose of IL-12 isbetween a range of about 22.5 μg/m² and about 52.5 μg/m².

In another aspect, the present invention provides methods for increasingsurvival in a subject, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration comprising theadministration of a low dose of IL-12 to the subject following an acuteexposure to non-therapeutic whole body ionizing radiation. In oneembodiment, the low dose of IL-12 is less than about 15 μg/m². Inanother embodiment, the low dose can be between about 3 μg/m² and about12 μg/m², such as 3 μg/m², 4 μg/m², 5 μg/m², 6 μg/m², 7 μg/m², 8 μg/m²,9 μg/m², 10 μg/m², 11 μg/m², and 12 μg/m² and all doses in-between. In aparticular embodiment, the preferred low dose of IL-12 is about 6 μg/m².

In one particular embodiment, a method is provided for increasingsurvival, and/or preserving bone marrow function, and/or promotinghematopoietic recovery or restoration in a human comprising theadministration of a low dose of IL-12 to said human following an acuteexposure to non-therapeutic whole body ionizing radiation. In a specificembodiment, the method for treating a human comprises administering alow dose of rhIL-12 in a range between about 1 hour and about 24 hoursafter acute exposure to whole body ionizing radiation, wherein the lowdose is less than about 400 ng/kg (15 μg/m²).

In another aspect, the present invention provides methods for increasingsurvival in a subject, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration comprising theadministration of an ultralow dose of IL-12 to the subject following anacute exposure to non-therapeutic whole body ionizing radiation. In oneembodiment, an ultralow dose of IL-12 used, such as a dose of less thanabout 3 μg/m². In another embodiment, the ultralow dose may be betweenabout 300 ng/m² and about 2400 ng/m², or between about 600 ng/m² andabout 1200 ng/m². In a particular embodiment, the low dose of IL-12 isabout 900 ng/m².

In still yet another aspect, the present invention provides a method forincreasing survival, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration in a human comprisingthe administration of an ultralow dose of IL-12 to the subject followingan acute exposure to non-therapeutic whole body ionizing radiation. In aspecific embodiment, the method for treating a human comprisesadministering a low dose of rhIL-12 in a range between about 1 hour andabout 24 hours after acute exposure to whole body ionizing radiation,wherein the low dose is less than about 80 ng/kg (3 μg/m²).

In certain embodiments of the invention, a low or ultralow dose of IL-12suitable for administration may be less than about 14 μg/m², or lessthan about 13 μg/m², 12 μg/m², 11 μg/m², 10 μg/m², 9 μg/m², 8 μg/m², 7μg/m², 6 μg/m², 5 μg/m², 4 μg/m², 3 μg/m², 2 μg/m², 1 μg/m², or lessthan about 900 ng/m², 800 ng/m², 700 ng/m², 600 ng/m², 500 ng/m², 400ng/m², 300 ng/m², 200 ng/m², or 100 ng/m².

It is well known that solutions of proteins that are formulated at lowconcentrations are susceptible to loss of a significant fraction of theprotein prior to administration. One major cause of this problem isadsorption of the protein on the sides of tubes, vials, syringes, andthe like. Accordingly, in certain aspects, when administered at low orultralow doses, it will be beneficial to administer IL-12 along with asuitable carrier molecule or bulking agent. In one embodiment, thecarrier agent may be a protein suitable for pharmaceuticaladministration, such as albumin. Generally, the carrier molecule orprotein will be present in the formulation in excess of IL-12 in orderto minimize the amount of IL-12 lost prior to administration. In certainembodiments, the carrier will be present at a concentration of at leastabout 2 times the concentration of IL-12, or at a concentration of atleast about 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, or more times theconcentration of IL-12 in the formulation.

Generally the IL-12 doses used in the methods for increasing survival ina subject, and/or preserving bone marrow function, and/or promotinghematopoietic recovery or restoration in a subject following an acuteexposure to non-therapeutic whole body ionizing radiation will be highenough to be effective for the treatment of a radiation syndrome, butlow enough to mitigate negative side effects associated with IL-12administrations, including for example, radiosensitivity of the GI tractand INF-γ up-regulation.

Advantageously, as provided by the methods of the present invention,administration of IL-12 may occur during any suitable time periodfollowing exposure to acute whole body radiation, up to and includingabout a week after exposure. Although the total dose of acute radiationwill factor into the time period in which IL-12 should be administered,according to one embodiment, IL-12 may be administered at any time up toabout 96 hours following exposure to radiation. In other embodiments,IL-12 can be administered at any time up to about 72 hourspost-irradiation, or at a time up to about 60 hours, 48 hours, 36 hours,24 hours, 18 hours, 12 hours, 8 hours, 6 hours, or less followingexposure to radiation.

In one specific embodiment, IL-12 is administered to a subject in needthereof between a range of about 1 hour to about 72 hours after exposureto ionizing radiation. In another embodiment, IL-12 is administeredbetween a range of about 1 hour and about 24 hours after exposure, orbetween a range of about 6 hours and about 24 hours following exposureto an acute dose of whole body ionizing radiation. Of great importancefor the usefulness of IL-12 in the face of a radiation disaster is thefact that IL-12 can be administered at protracted time points followingacute exposure to ionizing radiation. IL-12 is effective at any timepoint post radiation up to one week, but can provide especially higheffectiveness at time points up to 96 hours post radiation.

In certain other aspects, IL-12 can be administered at any reasonabletime point post radiation event, and be effective in increasingsurvival, and/or preserving bone marrow function, and/or promotinghematopoietic recovery. IL-12 administered at time points of up to 3hours, 6 hours, 24 hours, 48 hours and 72 hours are efficacious inincreasing survival, preserving bone marrow function, and promotinghematopoietic recovery. However, on one aspect, IL-12 acts on a residualsubpopulation of hematopoietic stem cells, and thus it is expected to beeffective at 96 hours, 120 hours and up to one week following acuteexposure to ionizing radiation.

In another aspect of the invention, methods are provided for increasingsurvival in a subject, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration comprising theadministration of more than one dose of IL-12 to a subject at protractedtimes following acute exposure to non-therapeutic whole body ionizingradiation. For example, in one embodiment, the method comprises theadministration of a first therapeutically effective dose of IL-12 at atime up to about 24 hours post-irradiation and a second therapeuticallyeffective dose of IL-12 at a second time up to about 72 hours after saidfirst dose. In a particular embodiment, the first IL-12 dose can beadministered between a range of about 1 hour and about 24 hourspost-irradiation, or between a range of about 6 hours and about 24 hourspost-irradiation, and said second IL-12 dose between a range of about 48hours and about 1 week post-irradiation, or between a range of about 48hours and about 94 hours post irradiation.

In one specific embodiment, the methods herein comprise administering afirst dose of less than about 75 μg/m² IL-12 to a subject at a timebetween about 1 hour and about 24 hours after acute exposure to wholebody radiation. In other embodiments, the method comprisesadministration of less than about 60 μg/m² IL-12, or less than about 45μg/m² IL-12, 30 μg/m² IL-12, 15 μg/m² IL-12, or less IL-12 at aneffective time after acute exposure to radiation.

In other embodiments, methods of multiple-dose IL-12 administration cancomprise the administration of a first low or ultralow dose of IL-12,for example, a first dose of less than about 15 μg/m², or less thanabout 14 μg/m², or less than about 13 μg/m², 12 μg/m², 11 μg/m², 10μg/m², 9 μg/m², 8 μg/m², 7 μg/m², 6 μg/m², 5 μg/m², 4 μg/m², 3 μg/m², 2μg/m², 1 μg/m², or less than about 900 ng/m², 800 ng/m², 700 ng/m², 600ng/m², 500 ng/m², 400 ng/m², 300 ng/m², 200 ng/m², or 100 ng/m².

When administered in multiple doses, i.e. two, three, four, or more, thefirst IL-12 dose and subsequent IL-12 dose(s) can be equivalent doses,or they can be different dose amounts. For example, in certainembodiments, subsequent dose(s) can be administered at about 90% of theinitial dose, or at about 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, 20%,10% or less of the original dose.

In one particular embodiment, a method is provided for increasingsurvival in a human, and/or preserving bone marrow function, and/orpromoting hematopoictic recovery or restoration, said method comprisingthe steps of administering a first dose of IL-12 to said human withinabout 24 hours following an acute exposure to non-therapeutic whole bodyionizing radiation, wherein said first dose is less than about 100 μg/m²and subsequently administering a second dose of IL-12 to said humanwithin about 96 hours following said acute exposure to non-therapeuticwhole body ionizing radiation, wherein said second dose is less thanabout 100 μg/m². In a specific embodiment, said second dose isadministered at least about 24 hours after said first dose isadministered. In another specific embodiment, said second dose is lessthan said first dose.

In yet other embodiments, methods are provided for increasing survivalin a subject, and/or preserving bone marrow function, and/or promotinghematopoietic recovery or restoration following an acute exposure tonon-therapeutic whole body ionizing radiation, comprising repeatedadministration of IL-12 for at least about a week, or at least about 2weeks, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks.In certain embodiments, the doses may be administered about once every12 hours, or about once every 24 hours, or about 1, 2, 3, 4, 5, 6, orseven times a week. In other embodiments, IL-12 may be administeredabout every other week or about 1, 2, 3, 4, 5, or more times a month. Insome embodiments, each IL-12 doses may be less than about 100 μg/m², oralternatively be an intermediate dose, a low dose, or an ultra low dose.In yet other embodiments, the doses may be decreased during the courseof a repeated administration.

Without being held to any particular theory, it is believed that whenthe hematopoietic system is compromised, as with acute whole bodyradiation, the IL-12 mediated pathway leading to the production of INF-γmay be sensitized. Consistent with this, Hixon et al. observed that whenIL-12 is administered to mice following whole body irradiation and bonemarrow transplant, INF-γ levels are greatly increased and acute lethaltoxicity of the gut results from the elevated levels of INF-γ. Theobserved acute lethal toxicity of the gut was dependant on INF-γ, asINF-γ knockout mice were resistant to IL-12 mediated toxicity. Theexperiments performed by Hixon et al. support a model in which IL-12administration results in an increase of INF-γ levels, and therefore anincrease in Crg-2 and Mig cytokine levels, resulting inanti-angiogenesis effects. Alternatively, but not mutually exclusivewith the above, by performing bone marrow transplants prior to IL-12administration, Hixon et al. provided INF-γ producing lymphocytes to themice, further increasing the potential for INF-γ production.

Thus, a possible mechanism for the decreased hematopoietic side effectsassociated with certain embodiments of the invention is that when arelatively low dose IL-12 is given to a mammal whose hematopoieticsystem is compromised, the dose is insufficient to upregulate INF-γproduction. Since INF-γ inhibits hematopoiesis and also appears to bethe major cytokine responsible for toxicity, the absence of INF-γupregulation upon administration of low and ultralow doses of IL-12, asused in the methods of the present invention, may be one of the factorsunderlying the discovery by the inventors that administration of low andultralow doses of IL-12 provides a hematopoietic protective and recoveryeffect without apparent toxicity.

Also of significance is the demonstration that exogenous administrationof IL-12 can expand long-term repopulating (LTR) hematopoietic stemcells (HSC) in vivo. Thus, without being bound by theory, HSC expansionby exogenous IL-12 can be the mechanism responsible for survival fromhematopoietic injury resulting from lethal radiation exposure at latertime points, e.g., 24 hours post-irradiation. Another potentialmechanism relates to the ability of IL-12 to induce DNA repair andreduce apoptosis in hematopoietic stem cells (HSC) following radiationexposure.

While most bone marrow progenitor and stem cells are susceptible to celldeath after high dose radiation, subpopulations of HSC or accessorycells are selectively more radioresistant, presumably because thesecells exist in a largely noncycling (G₀) state. In humans, theseradioresistant cells can play an important role in recovery ofhematopoiesis after exposure to doses as high as 6 Gy, albeit with areduced capacity for self-renewal in the absence of exogenous IL-12.

Another determinant for hematopoietic reconstitution is non-homogeneityof the radiation dose, which can spare some marrow sites that thenbecome the foci of hematopoietic activity. In either case, i.e., eitherthe residual presence of radioresistant HSC or inhomogeneity of theradiation dose, the present findings indicate that a subpopulation ofHSC marked by the presence of the IL-12 receptor (IL-12R+) survives andpersists after high dose radiation, and moreover, that this IL-12R+HSCsubpopulation is activated, expanded, and/or induced to repair itselfupon exogenous administration of IL-12.

Following radiation exposure, it has been discovered that IL-12 iseffective in mitigating the hematopoietic syndrome associated with acuteradiation syndrome. Specifically, embodiments of the present inventionprovide methods for increasing survival, and/or preserving bone marrowfunction, and/or promoting hematopoietic recovery by administering oneor more effective dose(s) of IL-12 to a subject following acute exposureto ionizing radiation.

For humans, as shown in Table 2, the early signs of hematopoieticsyndrome start to occur in the range of radiation doses of 2 Gy orgreater. Similarly, at radiation doses between about 5.5-7.5 Gy,pancytopenia and moderate GI damage occurs in humans. Advantageously,when administered according to the methods of the present invention,IL-12 is effective in alleviating the pancytopenia at these radiationdose levels, preserving bone marrow function and will not induce furtherGI damage. However, the radiation dose rate can also affect the relativelevel of radiation injury. Thus, two radiation doses given at twodifferent dose rates can show differences in the severity of therelative radiation injury.

TABLE 2 Phases of Radiation Injury* Dose Manifestation of PrognosisRange Gy Prodrome Illness (without Therapy) 0.5-1.0 Mild Slight decreasein Almost certain blood cell counts survival 1.0-2.0 Mild to Early signsof bone Highly probable moderate marrow damage survival (>90% ofvictims) 2.0-3.5 Moderate Moderate to severe Probable bone marrow damagesurvival 3.5-5.5 Severe Severe bone marrow Death within damage, slightGI 3.5-6 wk (50% damage of victims) 5.5-7.5 Severe Pancytopenia andDeath probable moderate GI damage within 2-3 wk  7.5-10.0 Severe MarkedGI and bone Death probable marrow damage, hypo- within 1-2.5 wk tension10.0-20.0 Severe Severe GI damage, Death certain pneuomonitis, alteredwithin 5-12 d mental status, cogni- tive dysfunction 20.0-30.0 SevereCerebrovascular col- Death certain lapse, fever, shock within 2-5 d*Modified from Walker R I, Cerveny R J, eds. (21), GI = gastrointestinal

For other mammals embraced by the methods and compositions of thepresent invention, for example mice, rats, guinea pigs, hamsters, cats,dogs, cattle, horses, sheep, pigs, rabbits, deer, monkeys, and the like,the radiation dose that can induce hematopoietic syndrome varies withthe species and strain. For example, for rhesus monkeys, the LD₅₀ isabout 7 Gy. For certain strains of mice, the LD₅₀ is also about 7 Gy,for example Balb-c mice. For other strains of mice, such as C57BL6, theLD₅₀ is about 7.5 Gy. The LD₅₀ can also exhibit differences based ongender or general health status of the animal.

Although it would be difficult to determine the exact extent ofradiation injury in a mammal exposed to acute ionizing radiationfollowing a radiation-related disaster, IL-12, when used in accordancewith embodiments of the present invention, will increase survival,and/or preserve bone marrow function, and/or promote hematopoicticrecovery of peripheral blood cell counts.

Accordingly, in some embodiments of the present invention, IL-12 isadministered to a subject that has been exposed to an acute dose ofionizing radiation of at least about 1.0 Gy, or an amount equivalent toan LD₁₀ in humans. In another embodiment, IL-12 is administered to asubject that has been exposed to about 3.5 Gy of ionizing radiation, ora dose equivalent to about LD₅₀ in humans. In yet other embodiments,IL-12 is useful for increasing survival, and/or preserving bone marrowfunction, and/or promoting hematopoietic recovery of peripheral bloodcell counts in a subject exposed to at least about 2.0 Gy, or at leastabout 3.0 Gy, 4.0 Gy, 5.0 Gy, 6.0 Gy, 7.0 Gy, 8.0 Gy, 9.0 Gy, 10.0 Gy,11.0 Gy, 12.0 Gy, 13.0 Gy, 14.0 Gy, 15.0 Gy, 20.0 Gy, 25.0 Gy, 30.0 Gy,or higher doses of acute ionizing radiation. Similarly, the dose ofionizing radiation can be expressed in terms of the percent lethal dose,for example, a dose equivalent of about LD₁, LD₅, LD₁₀, LD₂₀, LD₃₀,LD₄₀, LD₅₀, LD₆₀, LD₇₀, LD₈₀, LD₉₀, LD₉₅, LD₉₉, or LD₁₀₀.

A. Supportive Care

In another aspect of the invention, methods are provided for increasingsurvival in a subject, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration comprising theadministration of one or more dose of IL-12 to a subject at protractedtimes following acute exposure to non-therapeutic whole body ionizingradiation, wherein supportive care is given to said subjectsimultaneously or following administration of IL-12.

Supportive care modalities useful in conjunction with IL-12 fortreatment of a subject who has been exposed to an acute dose of wholebody ionizing radiation include, without limitation, administration offluids, one or more antibiotic, blood or blood component transfusions,administration of one or more growth factors or hematopoietic growthfactors, combination therapies and the like.

In one embodiment, supportive care comprises the administration of oneor more antibiotics. Antibiotic support can be any antibiotic that isuseful in preventing infections during periods of low blood cell countsincluding, without limitation, bactrim, ciprofloxacin, moxifloxacin, andthe like. Those of skill in the art will know of other antibioticsuseful for supportive care.

In another embodiment, supportive care comprises administration of oneor more growth factors, including hematopoietic growth factors. Manysuitable hematopoietic growth factors are known in the art including,without limitation, colony stimulating factors (CSF, G-CSF, GM-CSF,M-CSF, IL-3), erythropoietin, IL-1, IL-4, IL-5, IL-6, IL-7, IL-11, andthe like. Several FDA-approved hematopoietic growth factors arecurrently available, and thus may be used in the methods providedherein, such as G-CSF (Neupogen or Neulasta), IL-11, and erythropoietin(Epogen, Procrit or Aranesp). In some embodiments, supportive carecomprises the administration of keratinocyte growth factor (KGF orFGF7).

In one particular embodiment, erythropoietin administration can increasesurvival up to about 50% over and above that of IL-12 alone whensuper-lethal doses are used with no other supportive care measures, suchas antibiotic support or fluid administration. Super-lethal doses aredefined herein as radiation doses at or above 5.5 Gy. Erythropoietin isavailable as a FDA-approved recombinant protein drug for human use, suchas Epogen, Procrit or Aranesp. Generally, dosing with theseerythropoietin drugs will be simultaneous with or following theadministration of IL-12. Erythropoietin drugs can be repeated as needed,but generally not administered more than every other day, or every thirdday. Preferably erythropoietin is administered about 48 hours after thelast dose of IL-12.

An effective dose of erythropoietin for a human can be about 20 mg/kg,however, lower doses and higher doses are also effective in increasingsurvival when use as an adjuvant to IL-12 administration. Accordingly,in certain embodiments, erythropoietin is administered at about 1 mg/kg,or at about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more for treatment ina human, or at a dose equivalent amount for treatment in an animal otherthan a human.

In another embodiment, supportive care comprises the administration of ablood transfusion. As used herein, a blood transfusion may encompass awhole blood transfusion, or alternatively, transfusion of a bloodfraction or blood component, for example, a red blood cell transfusion,a platelet transfusion, a white blood cell transfusion. In a relatedembodiment, supportive care may comprise the administration of a bonemarrow or bone marrow stem cell transplant.

B. Formulations

IL-12 composition provided herein and used according to the methods ofthe invention can be formulated for administration via any known method,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. Further, anefficacious dose of IL-12 may differ with different routes ofadministration.

In certain embodiment IL-12 is administered following radiation exposureby intramuscular (IM) or subcutaneous (SC) routes. Advantageously, thesemodes of administration can be self-administered or administered by aperson with little or no medical training. As such, this aspect of theinvention allows for rapid responses under disaster conditions whereattention by those medical staff may be limited. Other modes ofadministration, however, such as intravenous and intraperitoneal, arealso compatible with the present invention.

The IL-12 compositions can be administered in a variety of unit dosageforms depending upon the method of administration. For example, unitdosage forms suitable for oral administration include, but are notlimited to, powder, tablets, pills, capsules and lozenges. It isrecognized that constructs when administered orally, should be protectedfrom digestion. This is typically accomplished either by complexing themolecules with a composition to render them resistant to acidic andenzymatic hydrolysis, or by packaging the molecules in an appropriatelyresistant carrier, such as a liposome or a protection barrier. Means ofprotecting agents from digestion are well known in the art.

In some embodiments, the formulations provided herein further compriseone or more pharmaceutically acceptable excipients, carriers, and/ordiluents. In addition, the formulations provided herein may furthercomprise other medicinal agents, carriers, adjuvants, diluents, tissuepermeation enhancers, solubilizers, and the like. Methods for preparingcompositions and formulations for pharmaceutical administration areknown to those skilled in the art (see, for example, REMINGTON'SPHARDMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, Pa.(1990)). Formulations used according to the methods of the invention mayinclude, for example, those taught in U.S. Pat. No. 5,744,132, which ishereby incorporated by reference in its entirety for all purposes.

C. Kits

In another aspect of the invention, kits are provided for increasingsurvival in a subject, and/or preserving bone marrow function, and/orpromoting hematopoietic recovery or restoration followingnon-therapeutic acute exposure to whole body ionizing radiation. In oneembodiment, a kit of the invention comprises one or more formulations ofa therapeutically effective amount of IL-12. In certain embodiments, thetherapeutically effective amount of IL-12 comprises a low dose of IL-12,for example, a dose that is less than about 15 μg/m². In otherembodiments, the therapeutically effective amount of IL-12 may comprisean ultralow dose of IL-12, for example, a dose that is less than about 3μg/m² In yet other embodiments, a kit as provided herein may comprisemultiple doses or formulations suitable for multi-dose administration ofIL-12 following radiation exposure.

In one specific embodiment, a kit is provided for increasing survival ina human, and/or preserving bone marrow function, and/or promotinghematopoietic recovery or restoration following non-therapeutic acuteexposure to whole body ionizing radiation, comprising a low dose orultralow dose of human IL-12. In one embodiment, the human IL-12 isrecombinant IL-12 or a recombinant IL-12 variant. In the kits of theinvention, IL-12 can be formulated so as to be delivered to a patientusing a wide variety of routes or modes of administration. In aparticular embodiment, IL-12 is formulated for injection in solution oras a lyophilized powder that can be easily reconstituted using sterilewater or a physiologically acceptable buffer.

In some embodiments, the kits of the invention may also comprise aformulation of a compound useful for supportive care of IL-12 treatmentfollowing radiation exposure. Suitable compounds include, withoutlimitation, antibiotics, for example, bactrim, ciprofloxacin, ormoxifloxacin, and hematopoietic growth factors, such as CSF, G-CSF,GM-CSF, M-CSF, IL-3, erythropoietin, erythropoietin-like molecules,IL-1, IL-4, IL-5, IL-6, IL-7, IL-11, and the like.

In certain embodiments, kits are provided for treating one or moreanimal following acute exposure to whole body ionizing radiation. Forexample, kits are provided for treating one or more of a human, dog,cat, guinea pig, hamster, cattle, horse, sheep, pig, rabbit, and thelike. In a particular embodiment, these kits contain one or more dose ofIL-12 specific for the animal to be treated. For example, a kit fortreating cattle may comprise bovine IL-12, or a kit for treating horsesmay comprise equine IL-12. In other embodiments, the kits comprise, forexample, murine or human IL-12 at a dose suitable for administration tothe particular animal to be treated.

In certain aspects, the kits provided herein are optionally housed in aradiation proof container (e.g., lead) or alternatively, the containerfor the kit is radiation resistant or proof (e.g., lead).

III. EXAMPLES Example 1 The IL-12-Facilitated Survival as a Function ofAdministration Schedule Following Super-Lethal Radiation (9 Gy)

This example illustrates the effects of administration time point ofIL-12 in a comparative experiment, where various time points weredirectly compared to assess relative survival and hematopoieticrecovery. IL-12 was administered at 3 hours after, 24 hours before, or24 hours before and 2 hours after (at two different doses of IL-12)using a 9 Gy radiation dose (unfractionated dose at a dose rate of 0.9Gy/min (γ-ray from cesium 137 irradiator). In this example, femaleC57BL/6 mice were used (13 weeks of age). All injections wereintravenous, and mice were started on antibiotics (Bactrim) followingradiation.

As shown in Table 3, survival data demonstrate that there was notsignificant differences among the various time points evaluated. Infact, administration of IL-12 at 3 hours after radiation or 24 hoursbefore radiation showed only a slight difference in survival, albeit thetwo groups were administered a different dose of IL-12. However, it wasdetermined that 100 ng (15 μg/m²) is the optimal dose of IL-12 whengiven 24 hours before lethal radiation. (See Table 3). Moreover, at thesame overall dose of IL-12 (200 ng/mouse; 30 μg/m²) given either in asplit dose of given at 24 hour before and 2 hours after radiation or asa single dose given at 3 hours after radiation, there was no differencein relative survival. These data suggest that a residual subpopulationof hematopoietic stem cells persist after radiation that can be actedupon by IL-12.

TABLE 3 Survival as a Function of Time of Administration of IL-12 at anAcute Radiation Dose of 9 Gy Time of Administration Dose of IL-12 %Survival PBS control 0 0 3 hours after radiation 200 ng (30 μg/m²) 60 24hour before radiation 100 ng (15 μg/m²) 50 24 hr before and 2 hr 100 ng,50 ng 70 after (15 μg/m², 7.5 μg/m²) 24 hr before and 2 hr 100 ng, 100ng 60 after (15 μg/m², 15 μg/m²)

Example 2 The IL-12-Facilitated Hematopoietic Recovery as a Function ofAdministration Schedule Following Super-Lethal Radiation (9 Gy)

The peripheral blood recovery for mice that survived the super-lethalradiation dose is shown in FIG. 1. Although there are some notabledifference in the neutrophil and red blood cell recovery, overall therecovery of these two blood cell groups was quite similar, and did notvary significantly as a function of the time point of IL-12administration. Platelet recovery, however, was significantly differentfor the 3 hour post- and 24 hour pre-radiation administration of IL-12,as compared to the split dose (pre-post) administration. For thisexample, mice were administered Bactrim in their drinking water, whichis known to induce thrombocytopenia. Mice were taken off antibiotics atabout 48 days, and subsequently, all groups showed an increase inplatelet counts. There results show that IL-12 can facilitate recoveryof all blood cell groups, including potent recovery of platelet counts,at super-lethal radiation dose (9 Gy TBI). Also notable is the length ofthe survival time. Mice were monitored for 57 days and were terminatedon day 60. All mice appeared in good health and had regained any lossesin body weight.

Example 3 IL-12-Facilitated Survival: Administration of IL-12 at 6 Hoursafter Lethal Radiation (LD_(100/10)) to Female Mice without AntibioticSupport

In this example, the effect of IL-12 when administered at 6 hours afteran unfractionated (acute), lethal dose of radiation (8 Gy) without theaddition of antibiotic support was evaluated.

A Kaplan-Meier plot of the data is shown in FIG. 2. Remarkably, at theLD_(100/10), 100% of the IL-12-treated mice at 6 hour post radiationsurvived. Moreover, the health status of the IL-12-treated mice wasremarkably stable during the period in which control mice experiencedthe effects of hematopoietic syndrome.

As shown in FIG. 3, although the IL-12-treated mice did lose about 14%,on average of their body weight by day 21 post radiation, they hadregained all of their lost body weight, and were slightly higher inweight than they were at the start of the experiment, by about day 30(19.1 g on day 0 vs. 19.6 g on day 30).

As would be expected, statistical analyses revealed that the survivaleffect was highly significant via the Mantel Chi-Square statisticalmethod (p=0.001), despite the small number of mice (n=5 for Group 1 andn=6 for Group 2). This example demonstrates the remarkable survivaleffects of IL-12 at 6 hours post radiation.

Example 4 IL-12-Facilitated Survival: Administration of IL-12 at 6 Hoursafter Lethal Radiation (LD_(100/21)) Using Male Mice and No AntibioticSupport

This example evaluated the effect of IL-12 when administered at 6 hoursafter an unfractionated (acute), lethal dose of radiation (8 Gy) in malemice without the addition of antibiotic support. This example shows alethal radiation survival study using male mice.

Two groups of mice were first exposed to a 7 Gy dose of radiation andeither treated with IL-12 or vehicle (PBS) at about 9 weeks of age. Forthe first round of radiation, Group 1 received PBS and Group 2 receivedIL-12, all mice survived following radiation. After 5 weeks, when it wasclear that all mice were healthy enough to undergo a second round ofradiation, the same mice were then subjected to a radiation dose of 8Gy. At this point, the mice were about 14 weeks of age and weighed 27grams on average. The IL-12 was adjusted according to the weight of themice. For the second round of radiation, Group 1 again received only PBSand Group 3 also received PBS, and Group 2 received IL-12 again.

A Kaplan-Meier plot of the data is shown in FIG. 4 (Groups 1 (PBS) vs. 2(IL-12)). Remarkably, it was found that after receiving an accumulateddose of 15 Gy, nearly 70% of the IL-12-treated mice survived (Group 2)when IL-12 was administered at 6 hours post radiation. Moreover, thehealth status of the IL-12-treated mice in Group 2 was observed to begood during the entire observation period for the surviving mice. Duringthe second round of radiation, surviving mice lost about 24% of theirbody weight by day 21 post radiation, and on day 30 these mice hadregained 10% of their body weight. These results are shown in FIG. 6.Remarkably, statistical analyses revealed that the survival effect wassignificant via the Chi-Square statistical Mantel method (p<0.05),despite the small number of mice (n=5 for Group 1 and n=6 for Group 2).

The data presented in FIGS. 3 and 4 were collected at the same time,i.e., the mice were all irradiated at the same time and received thesame radiation dose. However, there is clearly a notable difference inthe survival curve for control female mice as compared to control malemice. This may be due to apparent differences in intrinsic radiationrescue response for males and females, such as sex differences inendogenous IL-12 production following radiation or other relatedfactors.

Example 5 IL-12-Facilitated Survival: Administration of IL-12 at 24Hours after Lethal Radiation (LD_(90/30)) Using Female Mice and NoAntibiotic Support

In this example, IL-12 administration was evaluated when administered at24 hours after an unfractionated (acute), lethal dose of radiation (8Gy) without the addition of antibiotic support.

A Kaplan-Meier plot of the data is shown in FIG. 6. Remarkably, whenIL-12 was administered at 24 hours after an 8 Gy radiation dose, 90% ofthe IL-12 treated mice survived, whereas only 14% of the control micewere alive at 27 days post radiation. Moreover, the health status of theIL-12-treated mice was remarkably stable during the period in whichcontrol mice experienced the effects of hematopoietic syndrome.

As shown in FIG. 7, although the IL-12-treated mice did lose about 10%,on average, of their body weight by day 21 post radiation, they hadregained all of their lost body weight, and were slightly higher inweight than they were at the start of the experiment, by about day 27(18.5 g on day 0 vs. 19.2 g on day 27).

Statistical analyses revealed that the survival effect was significantvia the Mantel Chi-Square statistical method (p=0.001), despite thesmall number of mice (n=7 for Group 1 and n=8 for Group 2). This exampledemonstrates the remarkable survival effects of IL-12 at 24 hours postradiation.

Example 6 Low Dose Administration of IL-12 at 24 Hours and at 24 Hoursand 72 Hours after a Lethal Dose of Acute Whole Body Irradiation (LD₈₀)

160 female C57BL/6 mice in 16 groups with 10 mice per group wereirradiated with a lethal dose of acute whole body irradiation(LD_(80/30)) and then treated with IL-12 administration. Both the doseof IL-12 and the frequency of the dose (single vs. double dose) wereinvestigated. In the double dose experiment, the first dose of IL-12 wasadministered at 24 hours post radiation and the second dose of IL-12 wasadministered at 72 hours post radiation. For the single dose experiment,IL-12 was administered at 24 hours post radiation only. An outline ofthe study is shown in Table 4.

TABLE 4 Experimental design for single dose and double dose IL-12administration after acute whole body irradiation. muIL-12 dose muIL-12dose Group (ng per mouse) (μg/m²) 4A: Double Dose Experiment* 1 0(vehicle) 0 2 40 6 3 80 12 4 120 18 5 150 22.5 6 200 30 7 300 45 8 50075 4B: Single Dose Experiment* 1 0 (vehicle) 0 2 40 6 3 80 12 4 120 18 5150 22.5 6 200 30 7 300 45 8 500 75 *All injections were subcutaneous.

Mice treated with a double dose of IL-12 showed a linear dose responserelated to administration of mulL-12 at lethal radiation doses 24 and 72hours post radiation (FIG. 8A), whereas studies of single dosetreatments demonstrate a biphasic dose response related toadministration of muIL-12 at lethal radiation doses 24 hours postradiation (FIG. 8B). For the double dose experiment, higher doses ofmulL-12 gave better efficacy, as measured by survival following a LD₈₀radiation dose with the most efficacious dose being 300 ng (45 μg/m²)given twice. Interestingly, for the single dose experiments, the doseresponse curve was biphasic with 40 ng (6 μg/m²) and 200-300 ng (30μg/m²-45 μg/m²)) showing high efficacy at the same LD_(80/30) radiationdose. Average body weights for the surviving mice are shown in FIG. 9.

The efficacy at 300 ng (45 μg/m²) in the double dose experiment was 70%at the LD_(80/30) radiation dose (FIG. 8A). Notably, in the double doseexperiment, there were early deaths in many of the IL-12 treated groups,as compared to the control. Without being bound by theory, these earlydeaths may be due to protein aggregation resulting in immunogenicity.Consistent with this notion, there were very few early deaths in thesingle dose experiment. As can be seen via the statistical evaluationbelow, in the double dose experiment it appears that there may be twoeffects: 1) a highly therapeutic effect and 2) an effect that diminishesthe efficacy of the therapeutic effect.

The results of the single dose experiments show that a single low doseof IL-12 (40 ng; 6 μg/m²) administered at 24 hours after acute wholebody irradiation resulted in 90% survival at the LD_(80/30) (FIG. 8B).This single dose gave a 70% increase in survival as compared to thecontrol. Moreover, this increase in survival was highly statisticallysignificant (p<0.001; see statistical evaluation below).

For single dose experiments, The dose response curve was biphasic with asharp increase in survival at 40 ng (6 μg/m²), a dip in survival atintermediate doses and a second increase in survival time at dosesexceeding 200 ng (30 μg/m²; FIG. 14). Notably, weight loss was a highlysignificant predictor of lethality. Mice losing 20% or more of bodyweight did indeed subsequently die.

Without being bound by theory, the biphasic nature of the dose responsecurve (FIG. 14) may be explained by the presence of protein aggregatesin the administered IL-12. It is possible that at the 40 ng (6 μg/m²)dose, the least amount of aggregation is occurring, whereas in themiddle of the dose range the protein is more aggregated, and in thehigher dose range the protein is delivered as both an aggregated andnon-aggregated form. Consistently, if there are more aggregates leadingto some immunogenicity in the middle of the dose range, this mightexplain presence of a few early deaths, as compared to the control, inthe middle dose range. This type of aggregation and the resultantbiphasic dose response curve has been seen with other biologics.

Finally, it should be noted that the concentration actually delivered tothe mice in this example may be as low as 10-20% of what is indicated.This may be caused by the loss of IL-12 protein due to adsorption on thesides of tubes and syringes used in the study, as is common withsolutions of very low protein concentrations. These effects may bemitigated by various measures, including without limitation, the use ofcontainers with reduced affinity for the non-specific proteininteractions, or the pre-treatment of containers to reduce adsorption,as well as with the use of carrier molecules or proteins.

Statistical Analysis

FIG. 10 shows a stratified K-M analysis of the double dose experiments.In the double dosing experiments, doses of IL-12, 200-300 ng and above,increased survival in this experiment. Notably, when group 7 (300 ng; 45μg/m²) is compared to group 1 (0 ng, vehicle control), the effect wasstatistically significant (p<0.03; Tarone-Ware and Mantel methods; FIG.11). In contrast to the single dose experiments, significant low dosetherapeutic effects were not observed in the double dosing regimes.Weight loss was not a significant covariate in this experiment (FIG.9A), suggesting that the double dose protocol allowed for considerableweight loss without death, particularly at the highest IL-12 dose.

Stratified K-M analysis of the results generated in the single doseexperiments indicated an overall statistically significant effect ofIL-12 dose on survival time (p<0.02; Tarone-Ware method; FIG. 12).Strikingly, when groups 2 (40 ng; 6 μg/m²) and 6 (200 ng; 30 μg/m²) arecompared with group 1 (0 ng, vehicle control) in a three way K-Manalysis, the results are highly statistically significant (p<0.001;Tarone-Ware Method; FIG. 13).

Example 7 Determination of LD Values for Acute Whole Body Irradiation ofC57BL/6 Female Mice

Six groups of 10 female C57BL/6 mice each were subjected to increasingdoses of total body radiation using a Gammacell 40 irradiator withCesium source. The Specific dosage activity (Adjusted Dose Rate) for theirradiator was 5159 rad/hour. Groups were irradiated for the timeperiods shown in Table 5 in order to achieve the final dose of radiationas delineated in the table.

TABLE 5 Experimental design for acute exposure to whole bodyirradiation. Time Total Body Dose (min:sec) (Rad)  9:00 773.85  9:20802.22  9:40 831.45 10:00 859.83 10:20 888.21 10:40 917.44

The irradiated mice were housed for 30 days following irradiation. Themice were monitored daily for survival with the day of death recordedfor each mouse. The Kaplan-Meier graph of the results is presented inFIG. 15. Calculated radiation exposures for LD₃₀₍₃₀₎, LD₅₀₍₃₀₎, andLD₇₀₍₃₀₎ were about 782 rad (7.82 Gy), 788 rad (7.88 Gy), and 794 rad(7.94 Gy), respectively.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-27. (canceled)
 28. A kit for increasing survival in a subject, and/orpreserving bone marrow function, and/or promoting hematopoietic recoveryor restoration following non-therapeutic acute exposure to whole bodyionizing radiation, the kit comprising one or more formulations of atherapeutically effective dose of Interleukin-12 (IL-12).
 29. The kit ofclaim 28, wherein the kit is for increasing survival in a human subject,wherein the therapeutically effective dose of IL-12 is between fromabout 25.66 μg to about 1.71 μg.
 30. The kit of claim 28, wherein thekit is for increasing survival in a human subject, wherein thetherapeutically effective dose of IL-12 is less than about 17.11 μg. 31.The kit of claim 28, wherein the kit is for increasing survival in ahuman subject, wherein the therapeutically effective dose of IL-12 isless than about 13.68 μg.
 32. The kit of claim 28, wherein the IL-12 isformulated for intravenous, intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial, orintrathecal administration.
 33. The kit of claim 28, wherein the IL-12is formulated for injection in solution or as a lyophilized powder. 34.The kit of claim 28, wherein the IL-12 is recombinant IL-12.
 35. The kitof claim 34, wherein the IL-12 is recombinant human IL-12 (rhIL-12). 36.The kit of claim 38, wherein the rhIL-12 is HemaMax™.
 37. The kit ofclaim 28, wherein the IL-12 is modified so as to reduce theimmunogenicity of the protein.
 38. The kit of claim 37, wherein theIL-12 protein is modified with one or more water-soluble polymers. 39.The kit of claim 28, wherein the IL-12 is formulated in a tube, vial orsyringe.
 40. The kit of claim 28, wherein the IL-12 is administered witha carrier molecule or bulking agent.
 41. The kit of claim 40, whereinthe carrier molecule is an albumin.
 42. The kit of claim 40, wherein thecarrier molecule is present at a concentration of at least about twotimes the concentration of IL-12.
 43. The kit of claim 28, comprisingmultiple unit doses of IL-12.
 44. The kit of claim 28, furthercomprising a compound useful for supportive care of IL-12 treatmentfollowing radiation exposure selected from the group consisting ofantibiotics, hematopoietic growth factors, G-CSF, IL-3, erythropoietin,erythropoietin-like molecules, IL-1, IL-4, IL-5, IL-6, IL-7, IL-11, andany combination thereof.
 45. The kit of claim 28, wherein the IL-12 ishoused in a radiation proof or radiation resistant container.